Combined ablating agent based on spontaneous heating as well as preparation method and application of combined ablating agent

Abstract

The invention provides a combined ablating agent based on spontaneous heating and a preparation method and application thereof, and belongs to the technical field of medical materials. The LipoCA provided by the invention comprises a lipidosome, and CaO and silver nanoparticles which are wrapped in the lipidosome. In the invention, the lipidosome is used as a permeation barrier, CaO is wrapped in the lipidosome, the contact of CaO with H2O molecules and H < + > in a tumor microenvironment is delayed through a physical isolation effect, so that the exothermic reaction is accurately regulated and controlled, the Ag NPs can destroy a calcium metabolism steady state, induce Ca < 2 + > enrichment in tumors, mediate ROS production and promote cell apoptosis, and the Ag NPs, the CaO and the lipidosome have a synergistic effect, so that an exothermic effect is achieved. The composite material can improve the minimally invasive precise treatment of cancers, effectively avoids thermal burns of adjacent blood vessels and nerves, and has multiple functions of regulating and controlling the exothermic reaction rate, inhibiting thermal diffusion, inducing ROS (reactive oxygen species) generation and occluding blood vessels in tumors so as to enhance the thermal ablation curative effect and anti-tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a combined ablation agent based on self-heating, and a preparation method and application thereof. Background Art

[0002] Thermal ablation is a minimally invasive treatment technique that destroys diseased tissue through localized hyperthermia. Its core principle is to precisely destroy target tissue using focused energy while maximally preserving normal structures. It primarily includes radiofrequency ablation, microwave ablation, laser ablation, and high-intensity focused ultrasound ablation. Due to their minimal invasiveness, precision, and reproducibility, it is widely used in tumor treatment. However, regardless of the noninvasive or minimally invasive ablation technique used, all rely on exogenous energy input (such as electromagnetic waves, mechanical waves, or lasers) to generate localized hyperthermia to achieve tumor inactivation. These approaches have significant limitations in areas with dense blood vessels or adjacent to nerves. First, blood flow around the tumor creates a heat sink effect, continuously removing heat, making it difficult to maintain the target temperature at the effective therapeutic threshold, resulting in reduced thermal ablation efficiency. Second, unintended thermal fields generated by energy diffusion may exceed safety margins, causing the following damage: ① endothelial cell damage leading to thrombosis or vascular rupture; ② denaturation of nerve sheath proteins leading to paresthesia or motor dysfunction; and ③ thermal burns to adjacent hollow organs (such as the gastrointestinal tract and bile duct), leading to perforation or fistula formation. Therefore, developing a new type of thermal ablation device based on endogenous triggers of targets, metabolites or other protein molecules enriched in tumors is the key to improving minimally invasive and precise treatment of cancer and effectively avoiding thermal burns to adjacent blood vessels and nerves. Summary of the Invention

[0003] In view of this, the present invention aims to provide a combined ablative agent based on spontaneous heating, its preparation method, and its application. The present invention provides a novel combined ablative agent that can improve minimally invasive and precise cancer treatment and effectively avoid thermal burns to adjacent blood vessels and nerves.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a combined ablative agent, comprising liposomes and CaO and silver nanoparticles (Ag NPs) encapsulated in the liposomes.

[0006] Preferably, the mass ratio of the liposome, CaO and silver nanoparticles is 200-400:50-150:5-15.

[0007] Preferably, the particle size of the silver nanoparticles is 5 to 40 nm.

[0008] Preferably, the particle size of the CaO is 10 to 30 nm.

[0009] Preferably, the particle size of the combined ablation agent is 200-500 nm.

[0010] The present invention also provides a method for preparing the combined ablative agent described in the above technical solution, comprising the following steps:

[0011] mixing a phospholipid compound, cholesterol, polyethylene glycol (PEG), calcium oxide, silver nanoparticles and an organic solvent to obtain a dispersion;

[0012] The dispersion is evaporated to obtain the combined ablation agent.

[0013] Preferably, the phospholipid compound includes one or more of distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).

[0014] Preferably, the evaporation is reverse rotary evaporation, the temperature of the reverse rotary evaporation is 30-60° C., the vacuum degree is 50-150 mbar, and the rotation speed is 200-500 rpm.

[0015] The present invention also provides the use of the combined ablative agent described in the above technical solution or the combined ablative agent prepared by the preparation method described in the above technical solution in the preparation of a cancer thermal ablation therapeutic agent.

[0016] The present invention also provides a thermal ablation therapeutic agent (Lipo@CA+P, Lipo represents liposomes, CA represents CaO and silver nanoparticles, and P represents polydocanol foam sclerosant), including a thermal ablation agent and a polydocanol-based foam sclerosant (PFSA). The thermal ablation agent is the combined ablation agent described in the above technical scheme or the combined ablation agent prepared by the preparation method described in the above technical scheme.

[0017] The present invention provides a combined ablative agent (Lipo@CA), comprising liposomes and CaO and silver nanoparticles encapsulated in the liposomes.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a novel combined ablative agent that can improve minimally invasive and precise cancer treatment, effectively avoid thermal burns to adjacent blood vessels and nerves, and has multiple functions: regulating the rate of exothermic reactions, inhibiting heat diffusion, inducing reactive oxygen species (ROS) production, and occluding intratumoral blood vessels, thereby enhancing the efficacy of thermal ablation and anti-tumor treatment. The specific principles are as follows:

[0020] 1. Precise endogenously triggered thermal ablation

[0021] CaO is highly alkaline and hygroscopic, and reacts violently with water and releases a large amount of heat. The present invention uses liposomes to encapsulate CaO. After intratumoral injection, it enters cancer cells through membrane fusion. In the acidic microenvironment of the tumor, the liposome shell disintegrates and gradually releases CaO. CaO reacts with water and hydrogen ions (H + ) undergoes an exothermic reaction as follows:

[0022] CaO+H2O=Ca(OH)2

[0023] Ca(OH)2+2H + =Ca 2+ +2H2O

[0024] CaO + 2H + =Ca 2+ +H2O

[0025] This process consumes intratumoral H + , producing excess Ca 2+ , and releases a large amount of heat, causing the temperature inside the tumor to rise, destroying the cancer tissue, and achieving exothermic reaction-based thermal ablation therapy (ERTA). In addition, liposomes act as a permeability barrier, encapsulating CaO, and delaying its interaction with H2O molecules and H in the tumor microenvironment through physical isolation. + Contact, thereby achieving precise control of the exothermic reaction and avoiding the instantaneous high temperature rise caused by the traditional CaO exothermic reaction, which would damage the surrounding tissues.

[0026] 2. Highly effective synergistic anti-tumor effect

[0027] Biochemical effects on cancer cells. On the one hand, CaO decomposition releases heat to promote ERTA anti-tumor therapy, and also produces a large amount of Ca 2+ , destroying the calcium metabolism homeostasis in cancer cells, leading to Ca 2+ AgNPs and CaO were co-encapsulated in liposomes to obtain Lipo@CA. AgNPs can disrupt calcium metabolism homeostasis and induce Ca 2+ Enrichment, mediating ROS production, promoting cell apoptosis, the three synergistically mediate Ca 2+ The accumulation of CaO leads to the release of ROS. As the level of ROS increases, the mitochondrial membrane potential gradually decreases. On the other hand, CaO decomposes and digests H in the tumor microenvironment. + , which increases the pH value and the expression of apoptotic proteins (such as BCL-2), ultimately inhibiting the proliferation of cancer cells and promoting the inactivation and death of cancer cells.

[0028] The present invention also provides a method for preparing the combined ablative agent described in the above technical solution. The preparation method of the present invention is simple to operate and easy to realize industrial application.

[0029] Given that blood flow or spontaneous heat diffusion can cause heat loss, thereby affecting the temperature and efficiency of thermal ablation, the present invention also provides a thermal ablation therapeutic agent, including a thermal ablation agent and a polydocanol-based foam sclerosant. The thermal ablation therapeutic agent has a heat diffusion inhibitory effect. The principle is as follows: the polydocanol foam sclerosant, with its low thermal conductivity of air bubbles, prevents heat diffusion, promotes heat accumulation within the tumor and increases the ablation temperature, thereby enhancing the ERTA efficacy and reducing the risk of heat loss to surrounding normal tissues. At the same time, Lipo@CA+P with integrated PFSA synergistically promotes the expression of heat shock protein 70 (HSP70) and pro-apoptotic proteins (Caspase3 and Bax), and inhibits the expression of anti-apoptotic proteins (such as BCL-2), thereby inhibiting cancer cell proliferation and promoting the inactivation and death of cancer cells. PFSA also has a dual synergistic effect. PFSA not only inhibits heat diffusion and enhances the efficacy of ERTA, but is also a commonly used vascular occlusive agent in clinical practice, inducing endothelial cell damage and triggering thrombosis, ultimately leading to the closure of intratumoral vascular fibrosis, thereby cutting off the oxygen supply and nutritional support of the tumor tissue.

[0030] Moreover, the Lipo@CA and Lipo@CA+P of the present invention have good biosafety and clinical translation potential. Lipo@CA+P was injected into mouse tumors, and 16 days later, vital organs such as the heart, lungs, and kidneys were collected and stained with HE. No pathological changes such as necrosis and degeneration were found. When Lipo@CA was injected into the collected mouse blood, no severe hemolysis occurred even at a concentration of 500 μg / mL, which is far below the safe range. Mice treated with Lipo@CA+P ablation anti-tumor treatment had normal blood routine and liver and kidney function, with no significant changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the process for preparing a combined ablative agent according to Example 1 of the present invention;

[0032] Figure 2 Figure 3 shows the phagocytosis of Lipo@CA at the cellular level, where a is the flow cytometry (FCM) image, b is the confocal laser scanning microscopy (CLSM) image of Hepa1-6 cells after incubation with DiD-labeled Lipo@CA for different times, and c is the fluorescence intensity quantification.

[0033] Figure 3 Fluo-4AM calcium fluorescent probe staining was used to assess intracellular Ca 2+ Horizontal CLSM images;

[0034] Figure 4 Ca 2+ quantitative graphs;

[0035] Figure 5 The Ca content of Lipo@CA under different pH conditions 2+ Release circumstances;

[0036] Figure 6 is the change of pH intensity in different treatment groups;

[0037] Figure 7 CLSM images of cells in different treatment groups stained with BCECF-AM;

[0038] Figure 8 The exothermicity of CaO and the thermal diffusion inhibition of PFSA are shown in Figure 1, where a is CaO and Lipo@CA, and b is CaO+P and Lipo@CA+P.

[0039] Figure 9 The blood routine analysis of different treatment groups after treatment included WBC, RBC, HGB, HCT, MCV, MCH, MCHC and PLT;

[0040] Figure 10 (a) shows the CLSM images of Hepa1-6 cells stained with DCFH-DA fluorescence after different treatments, and (b) shows the quantification of ROS release.

[0041] Figure 11 Fluorescence microscopic image of mitochondrial membrane potential stained with JC-1;

[0042] Figure 12 CLSM images of Hepa1-6 cells after different treatments and calcein-AM and PI live-dead staining;

[0043] Figure 13 For quantitative assessment of cell viability;

[0044] Figure 14 In vivo anticancer evaluation of Lipo@CA+P, where a is a digital photo of tumors in Hepa 1-6 tumor-bearing mice in different treatment groups, b is the tumor weight in different treatment groups, and c is the survival rate of Hepa 1-6 tumor-bearing mice in different treatment groups over time;

[0045] Figure 15 The relative expression of various proteins in Hepa1-6 cells of different treatment groups was analyzed by Western blotting.

[0046] Figure 16a is the TEM image of CaO, b is the TEM image of Lipo@CA, c is the Zeta potential of Lipo@C and Lipo@CA in PFSA, d is the XPS spectrum of the typical Ca2p peak of CaO, e is the XPS spectrum of Lipo@CA, and f is the UV-visible absorption spectra of different treatment groups. DETAILED DESCRIPTION

[0047] The present invention provides a combined ablative agent, comprising liposomes and CaO and silver nanoparticles encapsulated in the liposomes.

[0048] In the present invention, the mass ratio of the liposomes, CaO and silver nanoparticles is preferably 200-400:50-150:5-15, specifically 300:100:10.

[0049] In the present invention, the particle size of the silver nanoparticles is preferably 5 to 40 nm, specifically 5, 10, 15, 20, 25, 30, 35 or 40 nm.

[0050] In the present invention, the particle size of the CaO is preferably 10 to 30 nm, specifically 10, 15, 20, 25 or 30 nm.

[0051] In the present invention, the particle size of the combined ablation agent is preferably 200 to 500 nm, specifically 200, 300, 400 or 500 nm.

[0052] In the present invention, the CaO reacts exothermically with water and hydrogen ions in the acidic tumor microenvironment to generate overloaded calcium ions and a large amount of endogenous heat, thereby inducing ERTA therapy, destroying blood vessels and cancer cells in the tumor, and blocking blood and oxygen supply. The overloaded Ca generated by the exothermic reaction of Ag NPs with CaO 2+ The liposome is a vesicle composed of a phospholipid bilayer, with its hydrophilic head facing outward and its hydrophobic tail facing inward, forming a natural barrier that wraps CaO and delays the reaction of CaO with H2O and H + Contact can limit the rate of exothermic reaction, prevent violent thermal explosion, and damage surrounding tissues, blood vessels and nerves. In addition, liposomes have the same structure as cell membranes and are highly accumulated in tumor cells through membrane fusion. However, when liposomes are in the acidic microenvironment of the tumor, the membrane structure disintegrates and gradually releases CaO, triggering an exothermic reaction and achieving ERTA of the tumor.

[0053] In the present invention, the combined ablation agent preferably decomposes and releases CaO and silver nanoparticles in a tumor microenvironment with a pH less than 6.5, thereby triggering thermal ablation.

[0054] The present invention also provides a method for preparing the combined ablative agent described in the above technical solution, comprising the following steps:

[0055] mixing phospholipid compounds, cholesterol, polyethylene glycol, calcium oxide, silver nanoparticles and an organic solvent to obtain a dispersion;

[0056] The dispersion is evaporated to obtain the combined ablation agent.

[0057] In the present invention, unless otherwise specified, the raw materials used are commercially available products in the art.

[0058] The invention mixes phospholipid compounds, cholesterol, polyethylene glycol, calcium oxide, silver nanoparticles and an organic solvent to obtain a dispersion.

[0059] In the present invention, the phospholipid compound preferably includes one or more of distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

[0060] In the present invention, the polyethylene glycol is preferably polyethylene glycol-2000.

[0061] In a specific embodiment of the present invention, the distearoylphosphatidylethanolamine and polyethylene glycol are preferably used in the form of distearoylphosphatidylethanolamine-mPEG2000 (DSPE-mPEG2000). In a specific embodiment of the present invention, the phospholipid compound preferably includes a mixture of dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine-mPEG2000 (DSPE-mPEG2000), wherein the mass ratio of DPPC to DSPE-mPEG2000 in the mixture is 1:2.

[0062] In the present invention, the phospholipid compound, cholesterol and polyethylene glycol are raw materials for preparing liposomes.

[0063] In the present invention, the organic solvent is preferably chloroform.

[0064] In a specific embodiment of the present invention, distearoylphosphatidylethanolamine-mPEG2000, dipalmitoylphosphatidylcholine, calcium oxide and cholesterol are added to chloroform, and then silver nanoparticles are added, and the mixture is stirred uniformly using a magnetic stirrer to obtain the dispersion.

[0065] After obtaining the dispersion, the present invention evaporates the dispersion to obtain the combined ablation agent.

[0066] In the present invention, the evaporation is preferably reverse rotary evaporation, the temperature of the reverse rotary evaporation is preferably 30 to 60°C, specifically 30, 40, 50 or 60°C, the vacuum degree is preferably 50 to 150 mbar, specifically 50, 100 or 150 mbar, and the rotation speed is preferably 200 to 500 rpm. There is no special limitation on the reverse rotary evaporation time of the present invention, as long as the organic solvent can be completely removed.

[0067] After the evaporation is completed, the present invention preferably sequentially hydrates, ultrasonicates and permeates the obtained lipid film to obtain the combined ablation agent.

[0068] In the present invention, the lipid membrane is preferably mixed with a Polidocanol solution, and the resulting liquid is sequentially passed through polycarbonate membranes with pore sizes of 500 nm and 200 nm to obtain the Lipo@CA.

[0069] In the present invention, the function of the Polidocanol solution is to be inserted between lipid films to form a double-layer liposome membrane structure.

[0070] In the present invention, the mass ratio of the lipid membrane to the polidocanol in the polidocanol solution is preferably 200-400:5-15, specifically 300:10.

[0071] In the present invention, the concentration of the polydocanol solution is preferably 1 to 3 mg / mL, specifically 1, 2 or 3 mg / mL.

[0072] In the present invention, the mixing is preferably performed by water bath sonication. The present invention has no particular limitation on the specific parameters of the water bath sonication, and methods well known to those skilled in the art may be used.

[0073] In the present invention, the number of times the liquid passes through a polycarbonate membrane with a pore size of 500 nm is preferably 20 times; the number of times the liquid passes through a polycarbonate membrane with a pore size of 200 nm is preferably 20 times.

[0074] The present invention also provides the use of the combined ablative agent described in the above technical solution or the combined ablative agent prepared by the preparation method described in the above technical solution in the preparation of a cancer thermal ablation therapeutic agent.

[0075] The present invention also provides a thermal ablation therapeutic agent, including a thermal ablation agent and a polydocanol-based foam hardener, wherein the thermal ablation agent is the combined ablation agent described in the above technical solution or the combined ablation agent prepared by the preparation method described in the above technical solution.

[0076] In the present invention, the polydocanol-based foam sclerosant, on the one hand, serves as a commonly used embolic agent in clinical practice, which can destroy blood vessels in the tumor, induce vascular occlusion, and block oxygen and blood supply; on the other hand, thanks to the low thermal conductivity of air (0.024-0.025 W / (m·K)), it can effectively inhibit spontaneous and blood flow-associated heat diffusion, promote heat accumulation in the tumor, and increase the ablation temperature. The thermal ablation agent and the polydocanol-based foam sclerosant synergistically promote the anti-tumor efficacy of ERTA.

[0077] In the present invention, the polydocanol-based foam hardener is preferably composed of a polydocanol solution and bubbles. The volume ratio of the polydocanol solution and the bubbles is preferably 3 to 5:1, specifically 3:1, 4:1 or 5:1. The mass volume percentage of the polydocanol solution is preferably 0.5% to 3%, specifically 0.5%, 1%, 2% or 3%.

[0078] In the present invention, the Lipo@CA and PFSA are preferably co-injected directly into the tumor tissue by intratumoral injection.

[0079] In the present invention, the mass ratio of Lipo@CA and PFSA is preferably 200-400:10-60, specifically 10:1, and the mass percentage of polydocanol in the PFSA is preferably 0.5%-3%, specifically 0.5%, 1%, 2% or 3%.

[0080] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0081] Example 1

[0082] 1. Preparation of Lipo@CA

[0083] Figure 1 This is a schematic diagram of the process for preparing a combined ablative agent according to Example 1 of the present invention.

[0084] First, distearoylphosphatidylethanolamine-mPEG2000, dipalmitoylphosphatidylcholine, calcium oxide, and cholesterol were dissolved in chloroform at a mass ratio of 20:10:10:1. Silver nanoparticles (calcium oxide to Ag NPs) were added (mass ratio of 100:10) and stirred uniformly using a magnetic stirrer. A lipid film was prepared by reverse rotary evaporation using a rotary evaporator at a vacuum of 100 mbar and a rotation speed of 200 rpm. Subsequently, 10 mL of polidocanol solution (concentration of 1 mg / mL) was added to the lipid film (0.3 g), and water-bath sonication was performed. Finally, the resulting solution was extruded 20 times through polycarbonate membranes with pore sizes of 500 nm and 200 nm, respectively, to obtain Lipo@CA. Lipo@CA consists of liposomes encapsulated with CaO and silver nanoparticles. The mass ratio of liposomes, CaO, and silver nanoparticles was 300:100:10.

[0085] 2. Usage

[0086] Lipo@CA (0.3 g) and PFSA (0.03 g, the mass percentage of polidocanol in PFSA is 3%) of the present invention were directly co-injected into the tumor tissue by intratumoral injection.

[0087] 3. Detection Methods

[0088] Characterization: Transmission electron microscopy (TEM), energy dispersive spectrometer (EDS), scanning transmission electron microscopy (STEM), ultraviolet spectrophotometer (UV3600), X-ray photoelectron spectroscopy (XPS), thermal infrared imaging, zeta potential and dynamic light scattering (DLS) were used to perform multi-dimensional characterization of the microstructure and morphological characteristics, elemental composition and spatial distribution, UV-visible optical absorption properties, element valence, temperature changes during the acidic buffer reaction, surface potential and hydrated particle size of this material to evaluate whether the prepared Lipo@CA successfully loaded CaO and Ag NPs, whether they were uniformly distributed and stable, and whether H+ triggered the decomposition of the structure and released heat, resulting in a temperature increase.

[0089] Figure 16 (a) TEM image of CaO (scale bar = 500 nm); (b) TEM image of Lipo@CA; (c) Zeta potential of Lipo@C and Lipo@CA in PFSA; (d) XPS spectrum of the typical Ca2p peak of CaO; (e) XPS spectrum of Lipo@CA; and (f) UV-Vis absorption spectra of different treatment groups. This indicates that CaO and Ag nanoparticles are successfully co-loaded in the Lipo@CA ablative agent. The negative charge of Lipo@C and Lipo@CA in PFSA facilitates high colloidal stability.

[0090] 4. Results

[0091] Lipo@CA basic characteristic parameters

[0092] 1. The diameter of Lipo@CA combined ablative agent is 200-500 nm.

[0093] 2. The Zeta potential of Lipo@CA in PFSA solution is -25mV, which is conducive to dispersion and accumulation in the tumor.

[0094] 3. Stability: The diameter and potential of Lipo@CA combined with ablative agent in PFSA showed no significant changes within 7 days.

[0095] 4. Safety: Even at a concentration of 500 μg / mL, the hemolysis rate of Lipo@CA combined with ablative agents is within a safe range. There is no significant damage to blood counts, liver and kidney function, or vital organs.

[0096] Comparative Example

[0097] Preparation of Lipo@C

[0098] The same as Example 1, except that AgNPs were omitted.

[0099] Lipo@CA's H + Trigger response and CaO exothermic reaction verification

[0100] When in the acidic microenvironment of the tumor, Lipo@CA enters the cancer cell through membrane fusion pathway, triggering the reaction of CaO and H + , H2O molecules undergo exothermic reaction, releasing a large amount of endogenous heat and Ca 2+ The specific experimental methods are as follows:

[0101] First, the phagocytic activity and distribution of the liposome nanoplatform Lipo@CA at the cellular level were examined. Lipo@CA was labeled with DiD fluorescent probe and co-cultured with mouse hepatocellular carcinoma cell line (Hepa1-6) for 0.5h, 1h, 2h, and 4h, respectively. The intracellular fluorescence level was then detected by confocal laser scanning microscopy (CLSM) and flow cytometry. Figure 2 Figure 3 shows the phagocytosis of Lipo@CA at the cellular level. (a) Flow cytometry (FCM) images show the phagocytosis of DiD-labeled Lipo@CA in Hepa1-6 cells after incubation for 0, 0.5, 1, 2, and 4 hours. (b) Confocal laser scanning microscopy (CLSM) images of Hepa1-6 cells incubated with DiD-labeled Lipo@CA for different times. (c) Fluorescence intensity quantification. The results showed that longer incubation time resulted in greater accumulation of Lipo@CA in Hepa1-6 cells.

[0102] Ca 2+Release amount determination. Hepa1-6 cells were incubated with PBS, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P, respectively. Then, the cells were stained with Fluo-4AM calcium fluorescent probe in the dark for 30 minutes. Finally, CLSM was used to detect the intracellular calcium ion level. Figures 3-4 Intracellular Ca 2+ Levels of assessment were assessed using Fluo-4AM (Ca 2+ Hepa1-6 cells stained with indicator) after being treated with different groups, Figure 3 Fluo-4AM calcium fluorescent probe staining was used to assess intracellular Ca 2+ Horizontal CLSM images, Figure 3 The scale bars are all 50 μm. Figure 4 Ca 2+ Quantitative graph, the results showed that the introduction of AgNPs and PFSA can induce Ca 2+ enriched, and the Ca in the Lipo@CA+P group 2+ The highest loading was in Hepa1-6 cells.

[0103] Lipo@CA's H + Trigger response verification. Lipo@CA was co-incubated with Hepa1-6 cells in culture medium with pH values ​​of 5.5, 6.5, and 7.4, respectively. 2+ Ca release assay 2+ level, Figure 5 The Ca content of Lipo@CA under different pH conditions 2+ It can be seen that as the pH value decreases, the Ca 2+ Release,Hepa1-6 cells were cultured in confocal culture dishes and divided into 7 groups: control, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P co-incubation. The cells were stained with BCECF-AM fluorescent probe, and finally the pH value was observed using CLSM. Figure 6 The changes in pH intensity in different treatment groups are shown in Figure 2. Figure 7 CLSM images of cells in different treatment groups stained with BCECF-AM. Figure 7 The scale bars are all 50 μm. The results show that the acid-base neutralization exothermic reaction of CaO is triggered in an acidic environment, resulting in Ca 2+ , pH value increased, H + reduce.

[0104] Exothermicity test and PFSA thermal diffusion inhibition test. When the solution pH is 5.5, thermal infrared imaging is used to monitor the temperature changes of CaO and Lipo@CA, as well as CaO+P and Lipo@CA+P groups. Figure 8 The graph shows the exothermicity of CaO and the thermal diffusion inhibition of PFSA. Figure 8 a is CaO and Lipo@CA, b is CaO+P and Lipo@CA+P. The results show that CaO directly reacts with H2O and H + The reaction generates a large amount of heat, causing a sudden temperature rise. However, after being encapsulated in liposomes, Lipo@CA and Lipo@CA+P exhibit a gradual temperature increase, effectively avoiding the uncontrollable and violent exothermic reaction-induced thermal explosion. Furthermore, the addition of PFSA inhibits heat diffusion and loss, resulting in a higher ablation temperature than without PFSA.

[0105] Biosafety Testing of Lipo@CA+P

[0106] 1) Evaluation of hemolysis rate. Mouse blood was collected in EP tubes containing EDTA (ethylenediaminetetraacetic acid, an anticoagulant) and centrifuged. ddH2O, PBS, and various concentrations of Lipo@CA were then added. Hemolysis was captured using a camera, and the absorbance of the samples was measured using a microplate reader. The results showed that even at a concentration of 500 μg / ml, the hemolysis rate of Lipo@CA remained within a safe range, with no significant changes.

[0107] 2) Toxicology Testing. Subcutaneous tumor-bearing mice were randomly divided into seven groups. 100 μL of PBS, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P were injected into the subcutaneous tumor tissue of each group. The mice's mental state, weight changes, diet, and activity were observed and recorded. Blood was drawn for routine blood tests and liver and kidney function tests. Sixteen days later, the mice were euthanized by cervical dislocation and dissected. The heart, liver, spleen, lungs, and kidneys were collected and stained with hematoxylin and eosin. Organ morphology was observed under a microscope to assess the presence of pathological changes such as degeneration, necrosis, and inflammation. Figure 9 Routine blood counts, including white blood cell counts (WBC), red blood cell counts (RBC), hematocrit (HGB), hematocrit (HCT), chromatin (MCV), chromatin choroidal choroid (MCH), chromatin choroidal ...

[0108] In vitro anticancer evaluation of Lipo@CA+P

[0109] 1) ROS level detection. The DCFH-DA fluorescent probe was used to evaluate the intracellular oxidative stress level and ROS generation induced by Lipo@CA+P. Hepa1-6 cells were divided into 7 groups and treated with PBS, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P, respectively. The DCFH-DA probe was added and the ROS generation was observed using CLSM. Figure 10 Figure a is the CLSM image of Hepa1-6 cells stained with DCFH-DA fluorescence after different treatments, where the scale bar is 50 μm. Figure b is the quantification of ROS release. The results showed that after the introduction of PFSA and AgNPs, the level of ROS production was significantly increased, so the ROS level in the Lipo@CA+P group was the highest.

[0110] 2) Mitochondrial membrane potential measurement. Hepa1-6 cells were treated with negative control, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P, respectively. JC-1 staining solution was added to the cells, and fluorescence changes were observed and recorded using a fluorescence microscope. Figure 11 The fluorescence microscopy images of mitochondrial membrane potential stained with JC-1 are shown in Figure 1. The scale bars are 100 μm. The results showed that the Ca generated by PFSA and AgNPs 2+ Overload promotes the production of ROS, reduces mitochondrial membrane potential, and promotes cell apoptosis.

[0111] 3) Cell apoptosis. After treatment (same as above), Hepa1-6 cells were resuspended and annexin V-FITC was added. After incubation at room temperature in the dark for 15 minutes, PI stain was added to the flow cytometry tube and flow cytometry was performed. Cell viability was assessed using calcein-AM and PI staining. Figure 12 CLSM images of Hepa1-6 cells after different treatments and calcein-AM and PI live-dead staining. Figure 12 The scale bars are all 100 μm. Figure 13 For the quantitative evaluation of cell viability, the results showed that the cells treated with Lipo@CA+P had the highest cell death rate and the lowest cell viability.

[0112] In vivo anticancer evaluation of Lipo@CA+P

[0113] Figure 14 In vivo anticancer evaluation of Lipo@CA+P, where a is the digital photo of tumors in Hepa 1-6 tumor-bearing mice in different treatment groups, b is the tumor weight in different treatment groups, and c is the survival rate of Hepa 1-6 tumor-bearing mice in different treatment groups over time.

[0114] 1) Hyperthermia Effect Testing. To further validate the thermal effects of ERTA and PFSA, Lipo@CA+P was injected directly into the tumor tissue of tumor-bearing mice. After anesthesia, the temperature changes in the tumor region were monitored using a photothermal imager. The results showed a similar temperature increase trend to that observed in vitro, further confirming that Lipo@CA+P can increase the temperature within the tumor and prevent heat spread.

[0115] 2) Detection of therapeutic effects. Tumor-bearing mice were randomly divided into 7 groups for in vivo anticancer treatment (n=4). Each group of mice received intratumoral injections of PBS, Ag, Lipo@C, Lipo@CA, PFSA, Lipo@C+P, and Lipo@CA+P. The body weight and tumor size of the mice were measured and recorded every two days, and the volume was calculated. After 16 days of treatment, the mice were euthanized, the tumors were surgically removed, and the tumors were photographed and weighed to observe changes in the tumors. The results showed that the Lipo@CA+P group had the best anti-tumor treatment effect.

[0116] Figure 15 WB analysis of the relative expression of various proteins in Hepa1-6 cells under different treatment groups showed that Lipo@CA (Lipo@CA+P) integrated with PFSA synergistically promoted the expression of heat shock protein 70 (HSP70) and pro-apoptotic proteins (Caspase3 and Bax), and inhibited the expression of anti-apoptotic proteins (such as BCL-2), ultimately achieving the purpose of inhibiting cancer cell proliferation and promoting the inactivation and death of cancer cells.

[0117] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation thereto. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

1. A combined ablative agent based on spontaneous heating, characterized in that: The invention comprises liposomes and CaO and silver nanoparticles encapsulated in the liposomes.

2. The combined ablation agent according to claim 1, characterized in that: The mass ratio of the liposome, CaO and silver nanoparticles is 200-400:50-150:5-15.

3. The combined ablation agent according to claim 1 or 2, characterized in that: The particle size of the silver nanoparticles is 5 to 40 nm.

4. The combined ablation agent according to claim 1 or 2, characterized in that: The particle size of the CaO is 10 to 30 nm.

5. The combined ablation agent according to claim 1, characterized in that: The particle size of the combined ablation agent is 200 to 500 nm.

6. The method for preparing the combined ablative agent according to any one of claims 1 to 5, characterized in that: The following steps are involved: mixing a phospholipid compound, cholesterol, polyethylene glycol, calcium oxide, silver nanoparticles and an organic solvent to obtain a precursor dispersion; The dispersion is evaporated to obtain the combined ablation agent.

7. The preparation method according to claim 6, characterized in that The phospholipid compound includes one or more of distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

8. The preparation method according to claim 6, characterized in that The evaporation is reverse rotary evaporation, the temperature of the reverse rotary evaporation is 30-60° C., the vacuum degree is 50-150 mbar, and the rotation speed is 200-500 rpm.

9. Use of the combined ablative agent according to any one of claims 1 to 5 or the combined ablative agent prepared by the preparation method according to any one of claims 6 to 8 in the preparation of a cancer thermal ablation therapeutic agent.

10. A thermal ablation therapeutic agent, characterized in that: It comprises a thermal ablative agent and a polydocanol-based foam hardener, wherein the thermal ablative agent is the combined ablative agent described in any one of claims 1 to 5 or the combined ablative agent prepared by the preparation method described in any one of claims 6 to 8.

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